Novel pharmacological therapy for neuronopathic gaucher disease

ABSTRACT

The instant disclosure relates to methods and compositions for the treatment of Gaucher disease, particularly type II and III neuronopathic Gaucher disease (nGD). The methods include the step of administering to an individual in need thereof an effective amount of a ryanodine receptor inhibitor or a pharmaceutically acceptable salt thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. 62/332,178,to Sun et al., filed on May 5, 2016, the contents of which areincorporated by reference in their entirety for all purposes.

BACKGROUND

Treatment of genetic brain disorders including neurodegenerativediseases are an unmet medical need. These include rare, lysosomalstorage diseases, and more common diseases such as Parkinson's disease.Neurodegenerative diseases strike about 50 million Americans each year,demanding enormous cost in medical expenses and lost productivity.Currently, there is no effective treatment for neurodegenerativediseases and there is a need for such in the art. The instant disclosureseeks to address one or more of the aforementioned needs in the art.

BRIEF SUMMARY

Disclosed are methods and compositions for the treatment of Gaucherdisease, particularly type II and III neuronopathic Gaucher disease(nGD). The methods include the step of administering to an individual inneed thereof an effective amount of a ryanodine receptor inhibitor or apharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Ryrs expression in 4L;C* mouse brains. (A) Decreased mRNAexpression (fold change of WT level) of three Ryrs in 4L;C* brainregions determined by RNASeq analysis (n=3 mice/group). (B) Immunoblotof brain lysate showed Ryr3 was expressed at normal levels at 13 days ofage and significantly decreased by 44 days of age in 4L;C* brain (27% ofWT level) compared to WT mice. P-values were from Student's t-test, n=3mice/group, 2-4 replicates of the experiment. (C) Immunofluorescencestaining of brain sections by anti-Ryr1 and anti-Ryr3 antibodies (green)showed decreased Ryr signals in 4L;C* brain including cortex andmidbrain regions. Scale bar is 20 μm for all the images. (D) Co-stainingof Ryr3 (green) with neural cell markers. NeuN (red) is for neurons.GFAP (red) is for astrocytes. 04 (red) is for oligodendrocytes. Ryr3signals were detected in neurons, astrocytes and oligodendrocytes in WTmidbrains. 4L;C* midbrain had low level of Ryr 3 signals in those cellscompared to WT. Scale bar is 20 μm for all the images. DAPI (blue)stained cell nuclei. Images are representative from n=3 mice for eachgenotype.

FIG. 2. Substrates, calcium levels and mitochondrial function in nGD(CBE-N2a) cells. (A) Substrates levels. GC and GS concentration wassignificantly increased in CBE-N2a cells compared to N2a cells.Treatment with G6 led to a significant reduction of GC and GS in N2a andCBE-N2a cells, respectively. Dantrolene alone did not significantlyaffect GC and GS levels in CBE-N2a cells. CBE-N2a cells treated withboth G6 and dantrolene showed similar level of GC and GS reduction to G6alone. (B) Cytosolic calcium levels. Differentiated N2a cells weretreated with CBE (2 mM) for 5 days. CBE-N2a cells were further incubatedfor 5 days with ryanodine (Ryan, 10 dantrolene (Dan, 12.5 AM) or G6 (0.8I.LM), or combinations of Dan/G6 or Ryan/G6. Cytosolic calcium level wasmeasured using Fura-2 as reporter after adding caffeine. Relativecytosolic calcium level [Fura-2 AF/F (340/380-510 nm)] normalized to mgprotein in cells shows the magnitude of cytosolic calcium above baseline(see method). CBE-N2a cells had significant higher cytosolic calciumlevels than N2a cells. Ryanodine, dantrolene, and G6 significantlyreduced cytosolic calcium levels in CBE-N2a cells. Combinations ofDan/G6 or Ryan/G6 in CBE-N2a cells also caused significant decrease incytosolic calcium levels. (C) Mitochondrial function in N2a cells.Reduced oxygen consumption rate (OCR, pmol/min/mg mitochondrial protein)parameters (ATP production, basal respiration and maximal respiration)in CBE-N2a cells were reduced compared to N2a cells. Dantrolene or G6treatment significantly improved OCR in CBE-N2a cells compared tountreated CBE-N2a cells. Relative levels of OCR in treated cellscompared to N2a were indicated below the graph. Each data are from 2 to4 experiments (n=3 cell or cell lysate samples/treatment group, sixreplicates/sample/experiment) and reported as mean±SEM. One-way ANOVAwith post-hoc Tukey test (P<0.05).

FIG. 3. Dantrolene treatment in 4L;C* mice. (A) Gait analysis. Leftstride (Left panel), right stride (Middle panel), base width (Rightpanel). The mice were subjected to two to three tests at 30 and 40 daysof age. Dantrolene (Dan) treatment significantly increased left andright strides in 4L;C* mice at 40 days of age, and reduced base width atboth 30 and 40 days of age compared to untreated 4L;C* mice. Littermate(4L;WT) mice that have no phenotype were used as normal controls in theanalysis. Data were analysed by Student's t-test. (B) Life span. Thesurvival rate of dantrolene treated 4L;C* mice (blue) was significantlyincreased compared to untreated 4L;C* mice (orange). Median survivaldays is 50 days or 44 days for treated or untreated 4L;C* mice,respectively. The life span of dantrolene treated 4L;C* mice wasprolonged by 12.3%. Littermate (4L;WT) control mice (black) had normallife span. Data are presented as Kaplan-Meier curve analysed byMantel-Cox test. (C) CNS-inflammation. Positive CD68 staining (brown) inmicroglial cells indicate inflammation in 4L;C* brain. Compared tountreated 4L;C, the CD68 signal was significantly reduced in dan-trolenetreated 4L;C* brains. The representative image for each group is shown.CD68 signal intensity in brain sections was quantitated by NIH image Jand presented as % of untreated 4L;C* level. P-value was from Student'st-test (n=2-3 mice/group). (D) Mitochondrial ATP production rate. 4L;Cbrain had 37% of ATP production rate (pmol/min/mg mitochondrial protein)compared to WT brains. Dantrolene treatment on 4L;C* mice improved ATPproduction to 77% of WT level. One-way ANOVA with post-hoc Tukey test(P<0.05), n=3 mice/group, 6 replicates/sample/assay, duplicate assays.(E) Immunoblot of LC3. LC3-II is barely detectable in WT brain. LC3-IIlevels were increased in 4L;C cerebrum compared to WT. Dantrolenetreated 4L;C* cerebrum showed significantly reduced level of LC3-IIcompared to untreated 4L;C*. One-way ANOVA with post-hoc Tukey test(P<0.05), n=3 mice/group, duplicate experiments.

FIG. 4. NeuN positive neurons in brain regions. WT, dantrolene (Dan)treated 4L;C* and untreated 4L;C* brain sagittal sections from 44 dayold mice were stained with anti-NeuN antibody. (A) Compared to WTcortex, cerebellum, midbrain and brain stem, 4L;C* mice had reduced NeuNpositive cells (green) in those regions. Representative images from eachgroup are shown. (B) Dantrolene treated 4L;C* mice had significantlymore NeuN positive cells than untreated 4L;C* in each region. In thegraph, NeuN positive cell counts in each group are shown as a percentageof WT for each brain region. Data were analysed by One-way ANOVA withpost-hoc Tukey test (P<0.05), n=4 images/section, 2 sections/mouse, 3mice/group.

FIG. 5. Ryr expression in dantrolene treated 4L;C* brain. (A) Immunoblotof Ryr3 in CBE-N2a cells. Ryr3 protein level was lower in CBE-N2a thanN2a cells, and increased in dantrolene treated CBE-N2a cells. (B) 4L;C*cerebrum showed significantly reduced Ryr3 protein at 9% of WT level. Indantrolene treated 4L;C* cerebrum, Ryr3 protein level was significantlyincreased compared to untreated 4L;C*. 4L;C* panel was spliced to makepanel layout consistent with other graphs. A dotted line shows splicearea. (C) Immunofluorescence staining of Ryr3.4L;C*midbrain and brainstem showed reduced Ryr3 (green) signal at 49% or 34% of WT level,respectively. In dantrolene treated 4L;C* brain, Ryr3 signal wasincreased to 94% in midbrain and 79% in brain stem of WT level. DAPI(blue) stained cell nuclei. Scale bar is 20 1.un for all the images. (Dand E) CAMK IV and calmodulin (CAM). 4L;C* cerebrum showed decreasedlevel of CAMK IV (D) and increased level of CAM (E) compared to WT.Dantrolene treatment normalized expression of CAMK IV and CAM to nearlyWT level One-way ANOVA with post-hoc Tukey test (P<0.05), n=2-3 mice,2-4 replicates of the experiment

FIG. 6. Scheme for generating nGD cell model. A) N2a cells weredifferentiated in the presence of retinoic acid (RA) and cAMP. Thedifferentiated N2a cells were treated with CBE, a Gcase inhibitor, togenerate nGD cells. B) Undifferentiated N2a Cells were stained positivewith anti-nestin antibody (green). The differentiated CBE-N2a cells werepositive for Map2 (red) a mature neuron marker. Scale bar is same forall images.

FIG. 7. Recording of cytosolic calcium levels. Differentiated N2a cellswere treated with CBE (2 mM) for 5 days. CBE-N2a cells were furtherincubated for 5 days with ryanodine (Ryan, 10 uM), dantrolene (Dan, 12.uM) or G6 (0.8 uM), or combinations of Dan/G6 or Ryan/G6. Cytosoliccalcium levels were measured using Fura-2 as a reporter. Baselinecalcium levels were measured prior to caffeine addition (−30 seconds).Cytosolic calcium levels at the baseline (0 second) are the following:(N2a/CBE)>(N2a/CBE/G6)>(N2a/CBE/Dan)˜(N2a/CBE/Dan/G6)>(N2a/CBE/Ryr/G6)>(N2a/CBE/Ryr)>(N2a).Calcium levels were recorded every 30 seconds for a duration of 300seconds after addition of caffeine. CBE-N2a cells treated showed reducedcytosolic calcium levels compared to CBE-N2a cells. Relative cytosoliccalcium level (Fura-2 ΔF/F (340/380-510 nm) for each time point werenormalized to mg of protein in the cells. A representative recording ofthree experiments is shown. Each data point repeated 3 times. Mean±SEMwas plotted in the graph.

FIG. 8. Gait analysis with mannitol-vehicle group. Left stride (Leftpanel), right stride (Middle panel), base width (Right panel). The micewere subjected to two to three tests at 30 and 40 days of age. Comparedto untreated 4L;C*, mannitol (Man)-4L;C* mice did not show changes inbase width. Dantrolene (Dan) treatment significantly reduced base widthat both 30 and 40 days of age compared to untreated 4L;C* and Man-4L;C*mice. Both Dan and Man improved left and right strides in 4L;C* mice at40 days of age, however, Dan-4L;C*mice (10%). Littermate (4L; WT) micethat have no phenotype were used as controls in the analysis. Data wereanalyzed by Student's t-test.

FIG. 9. NeuN positive neurons (green) in brain. (A) WT, dantrolenetreated 4L;C* and untreated 4L;c* brains were stained with anti-NeuNantibody. (B) In the graph, NeuN positive cells were quantitated fromimages of sagittal brain sections by Fiji for Image J and presented aspercentage of NeuN positive cells in WT brains. One-way ANOVA withpost-hoc Tukey test (p<0.05), (n=6 sections, 2 sections/mouse, 3mice/group). Scale bar is same for all images.

FIG. 10. (A) Increased GCase activity in dantrolene treated fibroblasts.The cells were treated with 12.5 uM dantrolene for 5 days. Dantrolenetreatment significantly increased GCase activity in WT, 4L/4L, 4L;C* and9H/9H fibroblasts, but did not affect GCase in 9V/9V fibroblasts.Student's t-test (n=3 cell lysate samples/genotype, each sample assayedin triplicates). (B) In dantrolene treated mice, brain GCase activitywas significantly increased compared to untreated mice at 44 days ofage. Student's t-test (n=6 mice/group). (C) Glucosylceramide (GC)concentration was decreased in dantrolene treated 4L;C* brain, but didnot reach significance, compared to the untreated 4L;C*.Glucosylsphingosine (GS) concentrations in the dantrolene treated 4L;C*brains were not significantly different from that in the untreated 4L;C*brain. Student's t-test (n=6 mice/group). (D) Co-staining of GCase(green) and Lamp1 (red). A bar graph is plotted at Y-axis of PearsonCorrelation Coefficient (PCC, r) calculated from the fluorescencesignals of captured images (n=8-13 cells/group) showing the degrees ofGCase co-localization with lysosomal marker Lamp 1. PCC was increased1.7-fold in dantrolene treated 4L;C* compared to untreated 4L;C* brain.One representative image per group is shown. One-way ANOVA with post-hocTukey test (p<0.05). Scale bar is same for all images. (E). GC (leftpanel) and GS (right panel) levels in 4L;C* brain with age. Student'st-test (n=2-10 mice per age group).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms and expressions used herein have the ordinary meaning as isaccorded to such terms and expressions with respect to theircorresponding respective areas of inquiry and study except wherespecific meanings have otherwise been set forth herein.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a method” includesa plurality of such methods and reference to “a dose” includes referenceto one or more doses and equivalents thereof known to those skilled inthe art, and so forth.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, or up to 10%, or up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value. Whereparticular values are described in the application and claims, unlessotherwise stated the term “about” meaning within an acceptable errorrange for the particular value should be assumed.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably to refer to an animal that is the object of treatment,observation and/or experiment. Generally, the term refers to a humanpatient, but the methods and compositions may be equally applicable tonon-human subjects such as other mammals. In some embodiments, the termsrefer to humans. In further embodiments, the terms may refer tochildren.

The term “therapeutically effective amount,” as used herein, refers toany amount of a compound which, as compared to a corresponding subjectwho has not received such amount, results in improved treatment,healing, prevention, or amelioration of a disease, disorder, or sideeffect, or a decrease in the rate of advancement of a disease ordisorder. The term also includes within its scope amounts effective toenhance normal physiological function.

The terms “treat,” “treating” or “treatment,” as used herein, refers tomethods of alleviating, abating or ameliorating a disease or conditionsymptoms, preventing additional symptoms, ameliorating or preventing theunderlying metabolic causes of symptoms, inhibiting the disease orcondition, arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orstopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

The term “pharmaceutically acceptable,” as used herein, refers amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compounds described herein.Such materials are administered to an individual without causingundesirable biological effects or interacting in a deleterious mannerwith any of the components of the composition in which it is contained.

The term “pharmaceutically acceptable salt,” as used herein, refers to aformulation of a compound that does not cause significant irritation toan organism to which it is administered and does not abrogate thebiological activity and properties of the compounds described herein.

The terms “composition” or “pharmaceutical composition,” as used herein,refers to a mixture of at least one compound, such as the compounds ofFormula (I) provided herein, with at least one and optionally more thanone other pharmaceutically acceptable chemical components, such ascarriers, stabilizers, diluents, dispersing agents, suspending agents,thickening agents, and/or excipients.

The term “carrier” applied to pharmaceutical compositions of thedisclosure refers to a diluent, excipient, or vehicle with which anactive compound (e.g., dextromethorphan) is administered. Suchpharmaceutical carriers can be sterile liquids, such as water, salinesolutions, aqueous dextrose solutions, aqueous glycerol solutions, andoils, including those of petroleum, animal, vegetable, or syntheticorigin, such as peanut oil, soybean oil, mineral oil, sesame oil and thelike. Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin, 18th Edition.

Disclosed are new treatment strategies for the management of Gaucherdisease, which may be similarly applied to treatment of otherneurodegenerative disorders such as Parkinson's disease. The disclosedapproaches include targeting the disease pathway, balancingglycosphingolipid substrate production and/or degradation, and restoringenzyme folding and trafficking to the lysosome by chaperones.

Gaucher Disease

Gaucher Disease is an autosomal recessively inherited lysosomal storagedisease (LSD), caused by mutations on the GBA1 locus. First reported byDr. Philippe Gaucher in 1882, the primary biochemical defect isenzymatic deficiency of lysosomal acid—β-glucosidase, and is the mostcommon lysosomal storage disease. 90% of LSDs are neuronopathic.

Gaucher disease is caused by mutations in GBA1 that encodes lysosomalacid 11-glucosidase (GCase) that has glucosylceramide (GC) and itsun-acylated form, glucosylsphingosine (GS) as substrates (1-3). Gaucherdisease is a common lysosomal storage disease with a frequency of1/57,000 live births (1). Based on neuronopathic involvement, Gaucherdisease is classified as type 1 (non-neuronopathic variant) and types 2and 3 (neuronopathic variants) (1). Type 2 patients present with acuteneurological signs and pathology within the first 3 to 6 months of lifeand with death before 2 years of age (1,4). Type 3 patients exhibitsub-acute neurological signs with a later onset and survival into the2nd to 4th decade (1,5,6). Two therapeutic strategies have shownclinical efficacy in treating non-neuronopathic Type 1 Gaucher diseaseand include: 1) enzyme replacement therapy (ERT) and 2) substratereduction therapy (SRT). However, the enzyme in ERT cannot cross theblood brain bather and the FDA approved SRT compounds, miglustat andeliglustat, do not show effective central nervous system (CNS) rescue(7-9). Thus, nGDs are not amenable to current ERT and SRT. Morerecently, pharmaceutical chaperones and newly developed small moleculesubstrate reduction agents have been shown to penetrate into the brain.However, these have limited efficacy in slowing disease progression andthey do not alter the disease course or prevent death in animal models(10-15). New therapeutic approaches are needed to protect neuronalfunction as a crucial goal for nGD intervention as has been a recentfocus to manage the CNS disease progression.

Accumulated substrates due to defective GCase function cause pathologyin the CNS of Gaucher disease. Studies from human patients, animalmodels and cell models show involvement of multiple pathologicalpathways in nGD pathogenesis including, inflammation, mitochondrialdysfunction, disrupted calcium homeostasis, altered autophagy/proteasefunction and necrosis (16-25). Disrupted calcium homeostasis, inparticular, is a major pathological factor contributing to manyneurodegenerative diseases and may lead to neurological deterioration inGD (18,19,25). Dantrolene is an antagonist of ryanodine receptors (Ryrs)and clinically used for the treatment of malignant hyperthermia andneuroleptic malignant syndrome (26). Ryrs are a class of intracellularcalcium channels, expressed in muscles, neurons and other cell typesthat mediate the release of calcium ions from intracellular organelles,sarcoplasmic reticulum and endoplasmic reticulum (ER). These areessential to a variety of signalling pathways (27). The unique mechanismof dantrolene in blocking intracellular calcium release through Ryrsmakes it an attractive potential approach to prevent neuronaldysfunction. Indeed, Applicant has discovered that potential clinicalutility for nGD may be possible in view of findings that modulatingcalcium with dantrolene improves neuronal function in severalneurodegenerative diseases including Huntington disease, Alzheimerdiseases and kinate-injury model (28-32).

There are three types of Gaucher Disease, as shown in Table 1.

TABLE 1 Types of Gaucher Disease Type 1 Type 2 Type 3 Non-neuronopathicAcute neuronopathic Sub-acute neuronopathic HepatosplenomegalyNeurodegeneration Neurodegeneration Bone Disease Visceral involvement6-80+ years <2 years 10-40 years 1/40,000 1/100,000 1/100,000 AshkenaziJews 1/850

95% are Type 1, 1% are Type 2, and 5% are Type 3. Approximately 400patients were reported to the International Collaborative GaucherRegistry (2015). Types 2 and 3 are neuronopathic.

Symptoms of neuronopathic Gaucher (nGD) disease include ataxia,dementia, seizures (progressive myoclonic), progressive spasticity,abnormal brainstem auditory evoked potentials; cognitive impairment andpathologic reflexes, dysphagia or apnea. Histologically, neuronal loss,lipid laden macrophages in periadventitial region, gliosis andmicroglial proliferation are observed. Biochemically, increased levelsof glucosylceramide and glucosylsphingosine in the brain are observed.GBA1 mutations confer a 20-30-fold increased risk for development ofParkinson's disease (PD). Approximately 7-10% of PD patients have a GBA1mutation, and GBA1 is listed as one of the target genes for diseasemodifying strategies for Parkinson's disease by The Michael J. FoxFoundation. The proposed mechanism of Gaucher disease linked Parkinson'sdisease. See Mazulli et al., Cell V146:37-52, 8 Jul. 2011.

Currently, clinical therapies for Gaucher disease include Enzymereplacement therapy (ERT) and Substrate reduction therapy (SRT). ERTtreatments include Cerezyme (imiglucerase) (Genzyme), VPRIV(velaglucerase alfa) (Shire), and ELELYSO (taliglucerase alfa) (Pfizer).SRT treatments include Cerdelga (eliglustat) (Genzyme), and Zavesca(miglustat) (Actelion). ERT increases Acid-beta-glucosidase (GCase),whereas SRT decreases glucosylceramide synthase (GCS).

Applicant has discovered a novel treatment method for Gaucher disease,particularly type II and III neuronopathic Gaucher disease (nGD).

In one aspect, the instant disclosure provides a method of treating anindividual having Gaucher disease (nGD), in particular type II or typeIII, comprising the step of administering an effective amount of aryanodine receptor inhibitor or a pharmaceutically acceptable saltthereof. In one aspect, the ryanodine receptor antagonist can be anyryanodine receptor antagonist known in the art.

In one aspect, the ryanodine receptor inhibitor may be selected fromdantrolene, JTV-519, Flecainide-d3, Flecainide,4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365), DHBP(1,1′-diheptyl-4,4′-bipyridium), Ruthenium red (R2751), Ryanodine, or acombination thereof.

JTV-519 is a potent inhibitor or the ryanodine receptor 2 (RYR2)blocker. RYR2 is a cardiac calcium channel that regulates calcium levelsin the sarcoplasmic reticulum. JTV-519 stabilizes RYR2 in the closedstate. (See, e.g., Science. 2004 Apr. 9; 304(5668):292-6. Protectionfrom cardiac arrhythmia through ryanodine receptor-stabilizing proteincalstabin2. Wehrens XH1, Lehnart S E, Reiken S R, Deng S X, Vest J A,Cervantes D, Coromilas J, Landry D W, Marks A R.)

Flecainide-d3 is a deuterated version of the antiarrythmic, Flecainide.(See, e.g., Mol Pharmacol. 2014 December; 86(6):696-706. doi:10.1124/mo1.114.094623. Epub 2014 Oct. 1. Multiple modes of ryanodinereceptor 2 inhibition by flecainide. Mehra D1, Imtiaz MS1, van HeldenDF1, Knollmann BC1, layer DR2.)

Flecainide is an antiarrythmic RyR-2 inhibitor (See, e.g., MolPharmacol. 2014 December; 86(6):696-706. doi: 10.1124/mo1.114.094623.Epub 2014 Oct. 1. Multiple modes of ryanodine receptor 2 inhibition byflecainide. Mehra D1, Imtiaz MS1, van Helden DF1, Knollmann BC1, layerDR2.)

4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365)—See, e.g.,Inhibition of Ryanodine Receptors by4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) in CaninePulmonary Arterial Smooth Muscle Cells, Olga Ostrovskaya, Ravi Goyal,Noah Osman, Claire E. McAllister, Isaac N. Pessah, Joseph R. Hume andSean M. Wilson, Journal of Pharmacology and Experimental TherapeuticsOctober 2007, 323 (1) 381-390; DOI:https://doi.org/10.1124/jpet.107.122119.

DHBP (1,1′-diheptyl-4,4′-bipyridium)—See, e.g., J Neurosci Res. 2007Aug. 1; 85(10):2207-15. Functional ryanodine receptors are expressed byhuman microglia and THP-1 cells: Their possible involvement inmodulation of neurotoxicity. Klegeris A1, Choi H B, McLarnon J G, McGeerP L

Additional ryanodine receptor antagonists include Ruthenium red (μM)(R2751), Ryanodine (>10 μM), Imperatoxin (nM) (I148), and Dantrolene(μM) (D9175), all of which are available from Sigma.

In one aspect, the ryanodine receptor inhibitor may be administered inan amount sufficient to reduce nGD associated autophagy. Reducedautophagy may be determined by reduced LC3-II levels as compared topre-treatment levels in an individual.

In one aspect, the ryanodine receptor may be administered in an amountsufficient to improve mitochondrial function.

In one aspect, the ryanodine receptor may be administered in an amountsufficient to improve sensory motor function.

In one aspect, the ryanodine receptor may be administered in an amountsufficient to increase or maintain Ryrs expression.

Further disclosed are methods of improving survival in an individualhaving nGD Type II or Type III. The method may comprise the step ofadministering an effective amount of a ryanodine receptor inhibitor.

Further disclosed is an article of manufacture. The article ofmanufacture may comprise a container comprising a label; and acomposition comprising a ryanodine receptor inhibitor or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier, wherein the label indicates that the composition forthe treatment of an individual having nGD Type II or Type III. Thearticle of manufacture may further comprise a means for delivery of saidcomposition to an individual in need thereof

Dosage

As will be apparent to those skilled in the art, dosages outside ofthese disclosed ranges may be administered in some cases. Further, it isnoted that the ordinary skilled clinician or treating physician willknow how and when to interrupt, adjust, or terminate therapy inconsideration of individual patient response.

In one aspect, the dosage of an agent disclosed herein, based on weightof the active compound, administered to an individual in need thereofmay be about 0.25 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, or more of a subject's body weight. Inanother embodiment, the dosage may be a unit dose of about 0.1 mg to 200mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg,0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg,1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5mg, or 1 mg to 2.5 mg.

In one aspect, an agent disclosed herein may be present in an amount offrom about 0.5% to about 95%, or from about 1% to about 90%, or fromabout 2% to about 85%, or from about 3% to about 80%, or from about 4%,about 75%, or from about 5% to about 70%, or from about 6%, about 65%,or from about 7% to about 60%, or from about 8% to about 55%, or fromabout 9% to about 50%, or from about 10% to about 40%, by weight of thecomposition.

The compositions may be administered in oral dosage forms such astablets, capsules (each of which includes sustained release or timedrelease formulations), pills, powders, granules, elixirs, tinctures,suspensions, syrups, and emulsions. They may also be administered inintravenous (bolus or infusion), intraperitoneal, subcutaneous, orintramuscular forms all utilizing dosage forms well known to those ofordinary skill in the pharmaceutical arts. The compositions may beadministered by intranasal route via topical use of suitable intranasalvehicles, or via a transdermal route, for example using conventionaltransdermal skin patches. A dosage protocol for administration using atransdermal delivery system may be continuous rather than intermittentthroughout the dosage regimen.

A dosage regimen will vary depending upon known factors such as thepharmacodynamic characteristics of the agents and their mode and routeof administration; the species, age, sex, health, medical condition, andweight of the patient, the nature and extent of the symptoms, the kindof concurrent treatment, the frequency of treatment, the route ofadministration, the renal and hepatic function of the patient, and thedesired effect. The effective amount of a drug required to prevent,counter, or arrest progression of a symptom or effect of Gaucher diseasecan be readily determined by an ordinarily skilled physician

Compositions may include suitable dosage forms for oral, parenteral(including subcutaneous, intramuscular, intradermal and intravenous),transdermal, sublingual, bronchial or nasal administration. Thus, if asolid carrier is used, the preparation may be tableted, placed in a hardgelatin capsule in powder or pellet form, or in the form of a troche orlozenge. The solid carrier may contain conventional excipients such asbinding agents, fillers, tableting lubricants, disintegrants, wettingagents and the like. The tablet may, if desired, be film coated byconventional techniques. Oral preparations include push-fit capsulesmade of gelatin, as well as soft, scaled capsules made of gelatin and acoating, such as glycerol or sorbitol. Push-fit capsules can containactive ingredients mixed with a filler or binders, such as lactose orstarches, lubricants, such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquid,or liquid polyethylene glycol with or without stabilizers. If a liquidcarrier is employed, the preparation may be in the form of a syrup,emulsion, soft gelatin capsule, sterile vehicle for injection, anaqueous or non-aqueous liquid suspension, or may be a dry product forreconstitution with water or other suitable vehicle before use. Liquidpreparations may contain conventional additives such as suspendingagents, emulsifying agents, wetting agents, non-aqueous vehicle(including edible oils), preservatives, as well as flavoring and/orcoloring agents. For parenteral administration, a vehicle normally willcomprise sterile water, at least in large part, although salinesolutions, glucose solutions and like may be utilized. Injectablesuspensions also may be used, in which case conventional suspendingagents may be employed. Conventional preservatives, buffering agents andthe like also may be added to the parenteral dosage forms. For topicalor nasal administration, penetrants or permeation agents that areappropriate to the particular barrier to be permeated are used in theformulation. Such penetrants are generally known in the art. Thepharmaceutical compositions are prepared by conventional techniquesappropriate to the desired preparation containing appropriate amounts ofthe active ingredient, that is, one or more of the disclosed activeagents or a pharmaceutically acceptable salt thereof according to theinvention.

The dosage of an agent disclosed herein used to achieve a therapeuticeffect will depend not only on such factors as the age, weight and sexof the patient and mode of administration, but also on the degree ofinhibition desired and the potency of an agent disclosed herein for theparticular disorder or disease concerned. It is also contemplated thatthe treatment and dosage of an agent disclosed herein may beadministered in unit dosage form and that the unit dosage form would beadjusted accordingly by one skilled in the art to reflect the relativelevel of activity. The decision as to the particular dosage to beemployed (and the number of times to be administered per day) is withinthe discretion of the physician, and may be varied by titration of thedosage to the particular circumstances of this invention to produce thedesired therapeutic effect.

Kits

Kits are also provided. In one aspect, a kit may comprise or consistessentially of agents or compositions described herein. The kit may be apackage that houses a container which may contain a compositioncomprising an oxime or pharmaceutically acceptable salt thereof asdisclosed herein, and also houses instructions for administering theagent or composition to a subject. In one aspect, a pharmaceutical packor kit is provided comprising one or more containers filled with one ormore composition as disclosed herein. Associated with such container(s)can be various written materials such as instructions for use, or anotice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use, orsale for human administration.

As there may be advantages to mixing a component of a compositiondescribed herein and a pharmaceutically acceptable carrier, excipient orvehicle near the time of use, kits in which components of thecompositions are packaged separately are disclosed. For example, the kitcan contain an active ingredient in a powdered or other dry form in, forexample, a sterile vial or ampule and, in a separate container withinthe kit, a carrier, excipient, or vehicle, or a component of a carrier,excipient, or vehicle (in liquid or dry form). In one aspect, the kitcan contain a component in a dry form, typically as a powder, often in alyophilized form in, for example, a sterile vial or ampule and, in aseparate container within the kit, a carrier, excipient, or vehicle, ora component of a carrier, excipient, or vehicle. Alternatively, the kitmay contain a component in the form of a concentrated solution that isdiluted prior to administration. Any of the components described herein,any of the carriers, excipients or vehicles described herein, and anycombination of components and carriers, excipients or vehicles can beincluded in a kit.

Optionally, a kit may also contain instructions for preparation or use(e.g., written instructions printed on the outer container or on aleaflet placed therein) and one or more devices to aid the preparationof the solution and/or its administration to a patient (e.g., one or aplurality of syringes, needles, filters, tape, tubing (e.g., tubing tofacilitate intravenous administration) alcohol swabs and/or theBand-Aid® applicator). Compositions which are more concentrated thanthose administered to a subject can be prepared. Accordingly, suchcompositions can be included in the kits with, optionally, suitablematerials (e.g., water, saline, or other physiologically acceptablesolutions) for dilution. Instructions included with the kit can include,where appropriate, instructions for dilution.

In other embodiments, the kits can include pre-mixed compositions andinstructions for solubilizing any precipitate that may have formedduring shipping or storage. Kits containing solutions of Compound I, ora pharmaceutically acceptable salt thereof, and one or more carriers,excipients or vehicles may also contain any of the materials mentionedabove (e.g., any device to aid in preparing the composition foradministration or in the administration per se). The instructions inthese kits may describe suitable indications (e.g., a description ofpatients amenable to treatment) and instructions for administering thesolution to a patient.

EXAMPLES

Neuronopathic Gaucher disease (nGD) manifests as severe neurologicalsymptoms in patients with no effective treatment available. Ryanodinereceptors (Ryrs) are a family of calcium release channels onintracellular stores. The following data was used to to determine ifRyrs are potential targets for nGD treatment. A nGD cell model (CBE-N2a)was created by inhibiting acid/3-glucosidase (GCase) in N2a cells withconduritol B epoxide (CBE). Enhanced cytosolic calcium in CBE-N2a cellswas blocked by either ryanodine or dantrolene, antagonists of Ryrs andby Genz-161, a glucosylceramide synthase inhibitor, suggestingsubstrate-mediated ER-calcium efflux occurs through ryanodine receptors.In the brain of a nGD (4L;C) mouse model, expression of Ryrs was normalat 13 days of age, but significantly decreased below the wild type levelin end-stage 4L;C* brains at 40 days. Treatment with dantrolene in 4L;C*mice starting at postnatal day 5 delayed neurological pathology andprolonged survival. Compared to untreated 4L;C* mice, dantrolenetreatment significantly improved gait, reduced LC3-II levels, improvedmitochondrial ATP production and reduced inflammation in the brain.Dantrolene treatment partially normalized Ryr expression and itspotential regulators, CAMK IV and calmodulin. Furthermore, dantrolenetreatment increased residual mutant GCase activity in 4L;C* brains.These data demonstrate that modulating Ryrs has neuroprotective effectsin nGD through mechanisms that protect the mitochondria, autophagy, Ryrexpression and enhance GCase activity. This study suggests that calciumsignalling stabilization, e.g. with dantrolene, could be a potentialdisease modifying therapy for nGD.

Here, nGD cell (CBE-N2a) and mouse (4L;C*) models were used to determinethe biochemical, histological, and behavioural effects of dantrolene innGD. The 4L;C* model is a viable analog of human nGD that developsprogressive accumulation of substrates and CNS pathology and symptoms(4,18,33,34). 4L;C* mice have been used to investigate pathologicalmechanisms and test potential therapeutics for nGD (14,18,35). Thepresent study shows dantrolene treatment improves mitochondrial functionand protects Ryrs expression in nGD cell and mouse models. Furthermore,dantrolene treatment improved gait, reduced inflammation and prolongedsurvival in 4L;C* mice, establishing therapeutic potential fordantrolene in nGD.

Results

Ryrs Expression in Brain of the nGD Mouse Model

The nGD mouse model (4L;C*) has the homozygous Gba1 mutation V394L/V394Land a lack of saposin C (33). The 4L;C* mice have substrate (GC and GS)accumulation in the brain that increases with age and is associated withprogressive gait impairment, brain pathology, and survival limited toapproximately 45 days (14,18,33). Transcriptome analysis by RNAseq ofcortex, brain stem, midbrain and cerebellum of 4L;C* mice brains showeda reduction of Ryr1, Ryr2 and Ryr3 mRNAs at 44 days of age (FIG. 1A)(18). Although all three Ryrs are expressed in the brain, Ryr2 ispredominantly seen in cardiac muscle (27,36). Levels of Ryrs protein inneuronal cells and mouse brains were determined for Ryr3 or Ryr1Immunoblot analysis showed Ryr3 protein in 4L;C* brains was maintainedat wild-type (WT) levels at 13 days of age, but was significantlyreduced to 27% of WT level at 44 days of age, the end-stage of thedisease (FIG. 1B). Immunofluorescence staining with anti-Ryr1 andanti-Ryr3 antibodies showed that Ryrs signal at 44-days in 4L;C* cortexand midbrain was reduced below WT levels (FIG. 1C). Neural cellsexpressing Ryrs were characterized by co-staining of anti-Ryr3 antibodywith either anti-NeuN (neuron), anti-GFAP (astro-cyte), or anti-04(oligodendrocyte) antibodies, respectively. The results showed that Ryr3was expressed in neurons, astrocytes and oligodendrocytes (FIG. 1D).4L;C* brains had reduced Ryr3 levels in all three cell types (FIG. 1D).These data demonstrate a reduction of Ryrs expression in 4L;C* brains atthe disease end-stage implicating Ryrs in nGD pathogenesis.

Dantrolene Blocks Substrate-Mediated Calcium Efflux from Ryrs in nGDCells

GC mediates ER calcium release (25). To determine if Ryrs are involvedin substrate-mediated ER calcium release, a new nGD cell model (CBE-N2a)was generated using N2a cells. N2a is a mouse neuroblastoma cell linethat can be differentiated into mature neurons by cAMP and retinoic acid(FIG. 6) (37). Differentiated N2a cells were treated with conduritol Bepoxide (CBE), an irreversible covalently bound GCase inhibitor. Thisresulted in significant GC and GS accumulation in CBE-N2a cells, therebycreating an nGD model (FIG. 2A). The effects of Genz-161 (G6), aninhibitor of glucosylceramide synthase to reduce the production of thesubstrates GC and GS, were evaluated in CBE-N2a cells (14). Treating N2aand CBE-N2a cells with G6 gave GC reductions to 7% and 26% of untreatedlevels, respectively (FIG. 2A). G6 treated CBE-N2a also hadsignificantly decreased GS (FIG. 2A). Dantrolene is an antagonist ofRyrs and blocks Ryr-mediated ER calcium release (17). Dantrolene alonedid not affect GC and GS levels in CBE-N2a cells (FIG. 2A). Co-treatmentof G6 and dantrolene of CBE-N2a cells led to a similar degree of GC andGS reduction as G6 alone, indicating a specific effect of G6 oninhibition of substrate accumulation (FIG. 2A). These resultsdemonstrate that G6 inhibits GC production and reduces substrateaccumulation in CBE-N2a cells.

Using CBE-N2a cells as an nGD cell model, the effects of dan-trolene,ryanodine and G6 on substrate-mediated ER-calcium release were evaluated(FIG. 2B). After CBE-N2a cells were treated with those agents, baselinecalcium levels were recorded prior caffeine addition. CBE-N2a cells havehigher calcium levels than in N2a cells without caffeine at baseline(FIG. 7). Caffeine is a stimulant or releaser of calcium from ER storesfor monitoring GC/GS-mediated ER-calcium levels and the Ryr response(38,39). After adding caffeine, cytosolic calcium levels were measuredevery 30 s over the duration of 300 s using Fura-2 (FIG. 7). Themagnitude of cytosolic calcium above baseline in the cells with eachtreatment is shown in FIG. 2B. CBE-N2a cells showed a significantincrease in cytosolic calcium levels compared to N2a cells. When CBE-N2acells were treated with G6, the calcium levels were significantly lowerthan that in untreated CBE-N2a cells (FIG. 2B), suggestingsubstrate-mediated calcium release. In dantrolene-treated CBE-N2a cells,calcium levels were significantly reduced compared to untreated CBE-N2aand approached levels observed in control N2a cells (FIG. 2B).Consistent with dantrolene, CBE-N2a cells treated with ryanodine alsoshowed a significant reduction in calcium levels (FIG. 2B). Co-treatmentof G6 and dantrolene or G6 and ryanodine also showed a significantdecrease in calcium levels. These results demonstrate thatsubstrate-mediated calcium release occurs through Ryrs.

Dantrolene Protects Mitochondrial Function in nGD Cells

To determine if substrate-mediated ER calcium efflux promotesmitochondrial dysfunction, the effects of dantrolene and G6 onmitochondrial function were evaluated in CBE-N2a cells. Treated cellswere plated on a Seahorse plate to assay mitochondrial function reportedas Oxygen Consumption Rate (OCR) that includes the following parameters:ATP production, basal respiration, and maximal respiration. CBE-N2acells showed a significant reduction in OCR as evidenced byapproximately 50% in all the parameters, including rate of ATPproduction, basal respiration, and maximal respiration, compared to N2acells, indicating reduced mitochondrial function in this nGD cell model(FIG. 2C). Dantrolene treatment significantly improved OCR in CBE-N2acells (FIG. 2C). G6 treatment also resulted in significant increases inOCR in CBE-N2a cells (FIG. 2C). The greatest increase in OCR wasachieved when CBE-N2a cells were treated with both dantrolene and G6(FIG. 2C). These results demonstrate that both reduction of substratelevels by G6 or antagonizing Ryrs by dantrolene have protective effectson mitochondrial function in nGD cells. Combining G6 and dantroleneresulted in more improvement than either dantrolene or G6 alone.

Dantrolene Treatment Mitigates the Neuropathic Phenotype in the nGDMouse Model

The in vivo efficacy of dantrolene on nGD was evaluated in the 4L;C*mouse model. 4L;C* mice were treated with dantrolene by intraperitoneal(IP) injection at 10 mg/kg on each of three days per week, starting frompostnatal day 5. Because dantrolene formulation contains mannitol, twocontrol groups were included in the study: untreated 4L;C* mice and4L;C* mice injected with mannitol at 30 mg/kg (same level of mannitol indantrolene formulation) on each of three days per week. Dantrolenetreated littermates (4L;WT) were also included as controls. Theselitter-mates do not show neurological impairments and live beyond 80days.

Gait was measured at 30 and 40 days of age. 4L;C* mice typically developa duck-like walking with paralysis of the hind limbs (18,33). Untreated4L;C* mice made significantly shorter strides and had a wider base widthat 30 and 40 days of age compared to 4L;WT littermates (FIG. 3A).Dantrolene treatment significantly reduced base stride width at 30 and40 days of age compared to age-matched untreated 4L;C* mice (FIG. 3A).

Dantrolene treated 4L;C* mice made significantly longer strides, a 30%increase compared to untreated 4L;C* mice at 40 days of age (FIG. 3A).However, the gait in dantrolene treated 4L;C* mice did begin todeteriorate after 50 days of age (data not shown). The life span ofdantrolene treated 4L;C* mice was also significantly extended comparedto untreated 4L;C* mice (P=0.0012) as determined by Kaplan-Meieranalyses and the Mantel-Cox test (FIG. 3B). The treatment prolongedsurvival in 4L;C* mice by 12.7% compared to untreated 4L;C* mice (FIG.3B). To determine if mannitol would affect the phenotype, one group of4L;C* mice was injected with mannitol and gait and survival wereevaluated. Mannitol-4L;C* mice showed no changes on base width, but theyhad a 10% increase (P<0.05) in stride length compared to untreated 4L;C*mice at 40 days of age. However, this improvement is significantly lessthan the 30% improvement in dantrolene treated 4L;C* mice (FIG. 8). Nodifferences were observed in life span between untreated andmannitol-treated 4L;C* mice (data not shown), indicating a specificeffect of dantrolene on survival. In the following biochemical andhistological studies, untreated 4L;C* brains were used as a control fordantrolene treatment effect. Dantrolene treatment did not affect body ororgan weights in treated mice. These results demonstrate that dantrolenesignificantly improved gait, delayed motor function decline, andextended the life span of 4L;C* mice.

CNS inflammation was determined by staining tissue sections with ananti-CD68 antibody. CD68 is a marker for activated microglia andmacrophages. Positive CD68 signals (brown) were detected in most brainregions of untreated 4L;C* mice (FIG. 3C). With dantrolene treatment,the CD68 signal showed a significant decrease of 63% of that inuntreated mice, indicating attenuation of CNS inflammation by dantrolene(FIG. 3C).

Brain mitochondrial function was determined by Seahorse assay usingisolated mitochondria from WT, untreated and dantrolene treated 4L;C*brains at 40 days of age. ATP production rate in 4L;C* brainmitochondria was significantly reduced compared to WT (FIG. 3D).Dantrolene treated 4L;C* mice showed significantly increasedmitochondrial ATP production rate in the brain, 77% of WT level comparedto 37% in the untreated 4L;C* brain (FIG. 3D) indicating a protection ofmitochondrial function in nGD by dantrolene.

Altered autophagy is also evident in nGD and 4L;C* mouse brains(20,33,34). LC3-II is a marker for autophagy activity and was evaluatedin dantrolene treated 4L;C* brains compared to untreated 4L;C* and WTbrains to determine the effect of the treatment on autophagy function.LC3-II is a membrane bound form of LC3. Elevated LC3-II levels indicatealtered autophagy. By immunoblot analysis, LC3-II was undetectable in WTbrain. Increased LC3-II levels were observed in untreated 4L;C* brainswhereas dantrolene treated 4L;C* brains had significantly reduced LC3-IIlevels at 50% of untreated level, but still more than WT level (FIG.3E). This result indicated that dantrolene treatment partially preventedalterations in autophagy in 4L;C* brains.

Neurodegeneration in 4L;C* brains were evaluated by counting NeuNpositive mature neurons in multiple brain regions (cortex, brain stem,midbrain and cerebellum) from whole sagittal sections (FIG. 4 and FIG.9). 4L;C* brains had significantly fewer NeuN positive cells (60-80% ofWT NeuN+ cells) than WT brains. Treatment with dantrolene resulted in anincrease in NeuN positive cells to >80% of WT NeuN+ cells. DecreasedNeuN positive cells were observed in cortex, cerebellum, midbrain andbrain stem of 4L;C* mice. All of those regions showed an increase inneurons with dantrolene treatment compared to the untreated 4L;C* mice(FIG. 4). These results indicated dantrolene treatment reducedneurodegeneration in 4L;C* mice.

Dantrolene Protects Ryr, Calmodulin and CAMK IV Expression

The effect of dantrolene on Ryr protein expression was determined in nGDcells and 4L;C* mouse brains (FIG. 5) Immunoblot analysis showed thatRyr3 protein was reduced to 47% of the WT level in CBE-N2a cells. Indantrolene-treated CBE-N2a cells, levels of Ryr3 were increased to 76%of WT level (FIG. 5A). In 4L;C* mice, Ryr3 was profoundly decreased tojust 9% of WT levels in the cerebrum at 44 days of age (FIG. 5B).Dantrolene treatment in 4L;C* mice significantly increased Ryr3 levelsto 77% of WT in the cerebrum (FIG. 5B) Immunofluorescence using ananti-Ryr3 antibody confirmed the increase in Ryr signals. In dantrolenetreated 4L;C* midbrain (94% of WT) and brain stem (79% of WT) regions(FIG. 5C). Ryr 3 signals were increased in neurons, astrocytes andoligodendrocytes in dantrolene treated 4L;C* mouse brains See Table 1.

TABLE 1 Ryr3 signal levels in brain cells Neuron AstrocyteOligodendrocyte WT ++++ +++ ++ 4L; C* + Dan ++ ++ + 4L; C⁺ + ± ±⁺relative level of immunoflourescence signal of Ryr3 in the mice brainco-stained with Ryr3 and NeuN for neuron, GFAP for astrocyte, or O4 foroligodendrocyte. Dan, dantrolene.

In the calcium/calmodulin-dependent protein kinase pathway, calmodulin,a calcium sensor protein, binds to calcium and promotes acalcium-dependent kinase activity including CAMK IV(Calcium/calmodulin-dependent protein kinase type IV) for geneexpression and biological functions (40). Calmodulin and CAMK IV arepotential modulators for Ryr expression. Decreased CAMK IV mRNA andincreased calmodulin 2 mRNA have been shown in 4L;C* brains using RNAseqanalyses (18). Expression levels of calmodulin and CAMK IV in 4L;C*brains were determined by immunoblot. CAMK IV protein level was reducedto 61% of WT level in untreated 4L;C* cerebrum. With dantrolenetreatment, the expression of CAMK IV was increased by 28% (FIG. 5, D).In this study, calmodulin protein was analysed using an anti-Camantibody that reacts to a class of calmodulins including calmodulin 2.Calmodulin levels were 2 fold above WT levels in 4L;C* brains and werenormalized to WT levels with dantrolene treatment (FIG. 5, E). Thesedata suggest that modulating cytosolic calcium levels with dantrolenehas protective effects on Ryrs expression as well as proteins involvedin the calcium-calmodulin dependent signalling pathway.

Dantrolene Treatment Effects on GCase Function

Modulating ER calcium release promotes chaperone enhancement of GCasestability and trafficking to the lysosome, thereby improving GCaseactivity (17,41,42). The effect of dantrolene on GCase activity wasevaluated in mouse fibroblast cells from Gba1 mutants homozygous forV493L/V394L (4L/4L), D409H/D409H (9H/9H), and D409V/D409V (9V/9V), 4L;C*and WT mice. Dantrolene-treated 4L/4L, 4L;C*, 9H/9H and WT fibroblastsshowed significantly increased GCase activity compared to the untreatedcells (FIG. 10, A). Dantrolene treated 9V/9V cells did not show anincrease in GCase activity.

This result showed that dantrolene promoted GCase activity in selectmutants, e.g. V394L and D409H, and WT enzyme.

In vivo effects of dantrolene on GCase function (activity and substratelevels) were analysed in dantrolene treated mouse brains at 44 days ofage. The cerebrum was used for GCase activity and cerebellum was usedfor substrates analysis. Compared to untreated 4L;C*, dantrolene-treated4L;C* cerebrum showed a 1.2 fold increase in GCase activity which issignificantly higher than untreated 4L;C* (FIG. 10, B). However,substrate (GC and GS) concentrations were not reduced in the treated4L;C* cerebellum (FIG. 10, C). Co-staining for GCase and Lamp1 of mousebrain sections showed a 1.7-fold increased colocalization of GCasewithin the lysosomes of dantrolene-treated 4L;C* mice compared tountreated 4L;C* brains. However, this level is <40% of Pearsoncoefficient for WT GCase (FIG. 10, D), indicating <40% mature mutantenzyme trafficked to the lysosomes in dantrolene-treated brains. Theseresults suggest that the effect of dantrolene on GCase activity with theregimen used in this study was not sufficient to translate intosignificant hydrolytic function for clearance of excess substrates.

DISCUSSION

Currently, there are no effective treatments available for nGD. The goalof the present study was to test the therapeutic potential of aryanodine receptor antagonist in cell and animal models of nGD.Applicant has shown that dantrolene treatment improves mitochondrialfunction, protects basal autophagy, delays abnormal gait, and mostimportantly prolongs survival in nGD mice. Dantrolene prevented thedecreased expression of Ryrs in nGD cells and mice, and normalizedexpression of calmodulin and CAMK IV, key molecules in thecalcium/calmodulin-dependent regulation pathway, in mice (40,43,44).Furthermore, dantrolene reduced inflammation and the loss of NeuN+neurons in mice indicating a compelling neuroprotective effect oftreatment. This study shows that modulating ER calcium efflux throughRyrs by dantrolene has therapeutic value for nGD.

Neuroprotection by dantrolene is likely through several mechanismsincluding protection of mitochondrial function, Ryrs expression andautophagy, and enhancement of GCase activity.

Abnormal calcium homeostasis has been implicated in GD (25,45,46).Accumulation of GC causes excess calcium efflux from ER, specificallythrough Ryrs in neurons in GD (38). Sustained increase in cytosoliccalcium accounts for the abnormal cellular function in many lysosomalstorage diseases including GD (47), although these changes are not fullyunderstood. Using CBE-N2a cells treated with G6 to inhibitglucosylceramide production, or dantrolene to inhibit calcium effluxthrough Ryrs, Applicant confirmed that increased cytosolic calciumefflux through Ryrs is mediated by excess substrates, supportingprevious findings (25). Most importantly, this new nGD cell model allowsfurther investigation of altered cellular function resulting fromdisrupted calcium homeostasis.

Ryr protein down-regulation in nGD cells and mouse brains is a novelfinding. All three Ryrs are expressed in brain. Ryr1 is predominantly inthe skeletal muscle and Ryr2 is mainly in cardiac muscle, whereas Ryr3is expressed at a low level in a variety of tissues including brain(27,36,48). Decreased expression of Ryrs has been reported in a mousemodel of Alzheimer disease that also shows aberrant cellular calciumhomeostasis (49). Ryrs protein levels in this study were determinedprimarily on Ryr3 with a high-quality antibody. Ryr3 was shown to beubiquitously expressed in the mouse brain. Reduced staining for Ryr3 wasfound in neurons, astrocytes and oligodendrocytes in 4L;C* mice atend-stage. Normal levels of Ryr3 protein were expressed in 4L;C* brainsat 13 days of age, but were reduced to 27% of WT level by the end stage(44 days), suggesting Ryr down-regulation is associated with diseaseprogression and increased substrate accumulation. Thus, Ryrdown-regulation is likely caused by substrate accumulation. In 4L;C*mouse brain, substrate levels were higher than that in WT even prior to13 days, and continued to increase with age (FIG. 10, E). Excesssubstrate can cause ER-calcium efflux and increases in cytosolic calcium(25). The sustained increase of cytosolic calcium could lead to afeed-back regulation, via calcium dependent transcription regulationpathways, and reduce the expression of Ryrs thereby preventing furthercalcium efflux from the ER. These results show that blocking ER-calciumrelease through Ryrs with dantrolene protects Ryr expression, supportingthis feed-back regulation mechanism. Early intervention before Ryr levelreduction would be critical to achieve maximal protection.

Expression of Ryrs can be regulated by the calcium-signalling pathway.In this pathway, calcium and calmodulin binding leads to basal CAMK IVactivity which can regulate the expression of Ryrs. Calmodulin bound tocalcium is required for the initiation of CAMK-CREB cascade for generegulation (43,44,50,51). Like many calcium binding proteins, Ryrs haveCREB binding elements (52), therefore, their expression is likelyregulated by CAMK IV-CREB signalling (53,54). Enhanced expression ofcalmodulin in 4L;C* brains may reflect increased cytosolic calcium.Reduced CAMK IV mRNA and protein levels in 4L;C* brains suggest thatless CAMK IV is available for gene regulation, or decreasedtranscriptional activity (18,52-54). Excess substrate-mediated calciumrelease in nGD brains could hinder calcium/calmodulin kinase signallingdependent CREB phosphorylation and lead to reduced Ryrs expression. WhennGD mice are treated at an earlier stage with dantrolene (starting atpostnatal 5 days) expression of CAMK IV and calmodulin were normalized,highlighting their involvement in the modulating Ryrs expression in nGD.Therefore, protection of Ryrs expression with dantrolene should benefitnGD.

Ryrs down-regulation was concomitant with disease progression.Importantly, the expression of Ryrs was protected by dantrolene. Thissuggests a role for Ryr in nGD pathogenesis and an attractive target fortherapy. Ryrs on ER are key for the regulation of intracellular calciumand an important calcium modulator for neuronal function (49,55). Bothryanodine and dantrolene are antagonists for Ryrs. Ryanodine is toxic tocells making it unsuitable for in vivo studies. In contrast, dantroleneis FDA approved for treatment of malignant hyperthermia (56), readilyavailable, and is distributed in tissues e.g. muscle and liver. It alsopenetrates the blood-brain barrier to allow for access into the brain(57). Dantrolene is specific to Ryr1 and Ryr3 (58). Blocking Ryrs withdantrolene is associated with improvement in learning and memoryperformance and protection of synaptic function in neurons in Alzheimerand Huntington disease models (28,32). The significant attenuation ofneuropathic phenotype in 4L;C* mice with dantrolene also suggests aclinical benefit for nGD. Impaired calcium homeostasis is a commonpathologic factor in glycosphingolipid storage diseases. Specificsubstrate accumulation causes reduced lysosomal calcium in Niemann-PickC1 disease and decreased ER calcium in Sandhoff and Niemann-Pick Adiseases (59-61). Thus, modulating Ryrs in order to normalize calciumhomeostasis could have therapeutic potential for glycosphingolipidstorage diseases.

Targeting Ryrs to modulate calcium homeostasis likely acts throughmultiple mechanisms to achieve neuroprotection in nGD, includingprotection of mitochondrial function and autophagy, normalizing geneexpression/regulation (Ryr), and rescuing mutant GCase activity.Mitochondrial dysfunction is a well-documented pathological feature innGD (18,62). Inhibition of GCase function by CBE leads to defectivemitochondrial function in a human dopaminergic cell line (23). Inchronic nGD mouse brains decreased mitochondrial ATP production andoxygen consumption are associated with protein aggregation (a-synucleinand amyloid precursor protein) on mitochondria (21). Impairedrespiratory chain and mitochondrial membrane potential has been reportedin primary neurons and astrocytes of acute nGD mice (23,62). Reducedmitochondrial function is also a significant feature in 4L;C* mice andin CBE-N2a cells (18). ER and mitochondria are physically andfunctionally linked with altered calcium homeostasis promotingmitochondrial dysfunction and disruption of the mitochondrial membranepotential (63-65). Furthermore, the role of Ryr in ER calcium andmitochondrial function is supported by a study of the conditionalknockout of cardiac Ryr2 in mice (66). Here, depletion of Ryr2 leads toreduced mitochondrial calcium and oxidative metabolism (66). Therefore,modulating calcium homeostasis through Ryrs would have benefits formitochondrial function. Indeed, in both CBE-N2a cells and 4L;C* mice,dantrolene treatment significantly improved the rate of mitochondrialoxygen consumption and ATP production. These data support the notionthat Ryr antagonism may have protective effects on mitochondrialfunction in nGD.

In many lysosomal storage diseases, defective lysosomal enzyme/proteinfunction interrupts the fusion of the lysosome with the autophagosomefor proper degradation of macromolecules (67-70). Abnormal autophagy,either increased or decreased, has been demonstrated in nGD cell andanimal models, and patient brain samples (5,20,22). Little is known,however, about the mechanisms regulating autophagy in nGD, although ithas been shown that autophagy in general is regulated by cytosoliccalcium (71,72). Increased calcium load induced by starvation is knownto induce autophagy (71). In this study, LC3-II was used to monitorautophagy. LC3 (microtubule-associated protein 1 light chain 3) has twoforms, LC3-I is distributed within the cytoplasm and nucleus and LC3-IIconjugates to phosphatidylethanolamine on the autophagosomal membrane.Increased LC3-II reflects an induction of autophagy in response tocellular stress. In the nGD mice, dantrolene treatment significantlyreduced LC3-II to WT levels suggesting that autophagy in nGD isregulated by the ER-calcium flux through Ryrs. Thus, modulatingcytosolic calcium levels could be a target for maintaining basalautophagy and normal cell function.

Modulation of ER-calcium can regulate calcium-dependent chaperones forGCase protein folding control and proper degradation (17,41,42,73).Inhibition of Ryrs reduces intraluminal calcium levels and enhances theexpression of ER chaperone proteins that promote protein folding.Specifically, for mutated GCase L′i44P in fibroblasts, this leads toincreased folding and trafficking to the lysosome (17,74). Modulating ERproteostasis by antagonizing Ryr with dantrolene has shown improvementin Niemann-Pick disease by enhanced type C1 protein level and attenuatedthe cholesterol and sphingolipids in Niemann-Pick type C diseasefibroblasts (73). In addition to Ryrs, blocking L type calcium channels(LTCC) also induces chaperone activity on mutant GCase (75). Althoughdiltiazem, an LTCC blocker, improves expression of cellular chaperonesand folding of the mutated enzymes in mouse and human fibroblasts(75,76), it fails to translate to similar changes in animal models of GD(76). In the present study, dantrolene was able to enhance GCaseactivity in select homozygous Gba1 mutations (V493L/V394L, D409H/D409H),4L;C* and WT mice fibroblasts. In 4L;C* mouse brains dantrolene enhancedmutant GCase activity and lysosomal localization, however, the level wasnot sufficient to prevent substrate accumulation. Increasingadministration to a daily regimen may achieve better efficacy on GCasehydrolytic function.

This study highlights a profound pathogenic role for Ryrs in nGD andsupports a novel strategy to preserve calcium homeostasis in the earlystages of nGD and thereby slow disease progression, and demonstratesthat targeting Ryrs to modulate calcium homeostasis has neuroprotectivepotential in nGD by protecting mitochondrial function, normalizingexpression of Ryrs, protecting autophagy, enhancing mutant GCaseactivity and reducing inflammation and neuron loss.

Materials and Methods

Materials

The following were from commercial sources: N2a (ATCCIO-CCLim-131, VA);CBE (EMD Millipore, Bedford, Mass.); Dantrolene, retinoic acid, dbcAMPand caffeine (Sigma, St. Louis, Mo.); M-PER Mammalian Protein ExtractionReagent, SuperSignalm West Dura ECL detection kit, Pierce BCA proteinassay kit (ThermoFisher Sci, Waltham, Mass.). Antibodies: Ryr1, Ryr3,Map2 and Nestin (EMD Millipore, Bedford, Mass.); CAM (calmodulin) (SantaCruz Biotechnology, Dallas, Tex.); LC3 (Novus, Littleton, Colo.); CAMKIV (calcium/calmodulin-dependent kinase IV) (Abcam, San Francisco,Calif.); CD68 (BD Biosciences, Franklin Lakes, N.J.); NuPAGE Gels (3-8%,4-12%, 10%, 16%) and Bis-Tris Buffer, Sodium Acetate Buffer, SeaBlueProtein Standard plus, PVDF membrane, iBlot transfer kit, and iBindCards (Life Technologies. Carlsbad, Calif.); AP colour reagent and APConjugate Substrate Kit (Bio-Red, Hercules, Calif.); VECTASHIELDmounting medium containing DAPI (Vector, Burlingame, Calif.); Glasschamber slides (LAB-TEK, Rochester, N.Y.). Genz-161(G6) was provided bySanofi Genzyme (Cambridge, Mass.).

Cell Culture and Treatment

N2a cells were maintained in DMEM/10% FBS (Gibco) medium. Neuronaldifferentiation of N2a was carried out in the differentiation mediumcontaining 0.5% FBS, 10 μM retinoic acid, 50 ng/ml GDNF (Alomore lab)and 1 mM dbcAMP in DMEM for 3 days (37). The differentiated N2a cellswere treated with 2 mM CBE for 5 days in the differentiation medium.Dantrolene (12.5 μM), ryanodine (10 μM) or G6 (0.8 μM) was added to theCBE-N2a cells and cultured for 5 days. The medium with the reagents waschanged every 2 days. The differentiation status of the neuronal cellswas confirmed by anti-Map2 (mature neuron) or anti-Nestin(undifferentiated neuronal cells) antibody staining. Cell viabilityafter each drug treatment was measured by CellTiter-Fluoirm CellViability assay (Promega). The dose for each compound was determinedfrom CellTiter-Fluor™ Cell Viability assay with >95% viability andendotoxin test of <0.05 EU/ml (GenScript ToxinSenor). The cells werefrom same passage and treated at the same time for all the experiments.

Calcium Level Measurement

Intracellular calcium levels were determined using the Fura-2 QBTCalcium Kit (Molecular Devices, Sunnyvale, Calif.) on 96 well plates byM5 SpectraMax plate reader (Molecular Devices). N2a cells were seeded ineach well (20,000/well) with differentiation medium (0.5% FBS, DMEM, 10AM retinoic acid, 1 mM dbcAMP) for 72 h (37). The differentiated N2acells were cultured with DMEM medium with 10% FBS containing CBE (2 mM)for 5 days, followed by treatment with different combinations ofdantrolene (12.5 μM), ryanodine (10 μM) and G6 (0.8 μM) for a further 5days. The cell medium was replaced every two days. The treated cellswere incubated with Fura-2 QBT dye at 37 C for 1 h for Fura-2 QBT uptake(Fura2 QBT® calcium kit protocol, Molecular Devices). Caffeine (10 mM)was used as an ER calcium efflux stimulant in the assay (38,39). Theplate reader setting was 340/380 nm (excitation) and 510 nm (emission).Baseline calcium levels were measured prior to caffeine addition (−30s). Cytosolic calcium levels were recorded every 30 s for duration of300 s after addition of caffeine. Relative cytosolic calcium levels weredetermined with the values at 300 s endpoint minus Os baseline. Relativecytosolic calcium level [Fura-2 AF/F (340/380510 nm] for control ortreated cells was calculated by Fura-2 fluorescence unit (FU) measuredat 340 nm/510 nm (AF=RFU/340 at 300 s end point minus baseline RFU/340at 0 s) divided by FU at 380 nm/510 nm (F=RFU/380 at 300 s-baselineRFU/380 at 0 s) (Fura2 QBT® calcium kit protocol, Molecular Devices) andnormalized to protein level in cells. Data were collected and analysedby SoftMax Pro software (Molecular Devices) and graphical charts weregenerated by Graphpad Prism 6.0.

Mice and Treatment

4L;C* mice were generated by backcrossing of V394L/V394L Gba1 (4L) andsaposin C−/− (C) homozygosity as described previously (33). The strainbackground of 4L;C* mice was C57BL/6J:129SvEv. Strain- and age-matchedWT mice and non-4L;C* littermates (no neurological phenotype) wereincluded as controls. All mice were housed under pathogen-freeconditions in an animal facility according to IACUC approved protocolsat Cincinnati Children's Hospital Medical Center (CCHMC).

The 4L;C* mice and non-4L;C* littermates were treated with Dantrolenesodium (Revonto® US WorldMeds, Louisville, Ky.) starting at postnatalday 5 by IP injection at 10 mg/kg, three days per week. Mouse bodyweight was recorded before each injection. Control mice received vehicle(mannitol, APP Pharmaceuticals, Schaumburg, Ill.) injections or noinjection at all. The mice were monitored for survival and gaitimpairments during the treatment.

Gait Analysis

Progression of the neurobehavioural phenotype was assessed by gaitanalysis to determine sensorimotor function (77,78). Mice were trainedto walk through a narrow alley leading into their home-cage. Oncetrained, paper was placed along the alley floor and the hind paws ofeach mouse were brushed with nontoxic paint. The mice were then placedat the beginning of the alley. As they walked into their home-cage theyleft their paw prints on the paper underneath. Stride length and widthwere determined by measuring the distance between hind paw prints. The4L;C* and control mice, untreated and non-4L;C* littermates were testedfor gait at 30 and 40 days of age.

RNAseq Analyses

Expression of Ryr mRNAs in 4L;C* brain regions (cortex, cerebellum,midbrain and brain stem) was analysed by RNAseq as described previously(18).

Immunohistochemistry and Immunofluorescence

Mouse brains were collected after transcardial perfusion with saline.Half of the brain (sagittal cut) was fixed in 4% paraformaldehyde (PFA)for processing as frozen blocks. CD68 monoclonal antibody staining wasperformed as previously described (33) using The BenchMark XT IHC/ISHStaining Module (Ventana Medical System, Tucson, Ariz.) at CCHMCPathology Research Core. Fixed sections were counterstained withhematoxylin. The sections were then scanned by AperiolmageScope v2. TheCD68 signal was quantified using Image J FIJI (79).

Immunofluorescence staining was performed on PFA fixed brains. The brainsections were incubated in 0.3% Triton X-100 for 30 min, and treatedwith 50 mM NH₄Cl in 1×PBS for 15 min followed by 1×PBS wash. Thesections were blocked for 1 hr at RT in Blocking buffer (10% goat serumand 0.4% Triton X-100 in PBS). Rabbit anti-Ryr 1 antibody (1:100) wasdiluted in the blocking solution and incubated overnight at 4° C. Afterwashing in PBS (3×10 min), the secondary antibody goat anti-rabbitconjugated with Alexa Fluor® 488 (1:1500) in blocking buffer was appliedto the sections and incubated for 2 hrs at RT. Mouse anti-Ryr 3 antibody(1:100) and goat anti-mouse conjugated with Alexa Fluor® 488 (1:1500)were used for Ryr 3 detection. For co-staining of neural cells withRyr3, mouse anti-NeuN antibody (1:500, Millipore), mouse anti-GFAP(1:100, Millipore) or mouse anti-04 (1:100, Millipore) with goatanti-mouse conjugated Alexa Fluor® 594 (1:1000) were used for NeuN,GFAP, or 04 detection, respectively. Rabbit anti-Ryr 3 (1:250,Millipore) with goat anti-rabbit conjugated Alexa Fluor® 488 (1:1000)were used for Ryr3. Lysosomal localization of GCase was determined usingrabbit anti-mouse GCase (1:50) antibody (15) with goat anti-rabbitconjugated Alexa Fluor® 488 (1:1000) for mouse GCase detection. Ratanti-Lamp1 (1:100, RDI) and rabbit anti-rat IgG conjugated Texas Red(1:1000, Abcam) were used for Lamp1 detection. Sections were mountedwith VECTASHIELD mounting medium containing DAPI (Vector H1200).Fluorescence signals were visualized and captured by Zeiss Axiovert 200M microscopy equipped with an Apotome. Pearson correlation coefficientsoftware is incorporated in Apotome microscope and used to analyse theco-localization of two signals (GCase and Lamp1) in cells. The averagePearson correlation coefficient number (from 0 to 1) was derived fromthe cells in multiple images (21). For counting NeuN⁺ neurons, mouseanti-NeuN antibody (1:500, Millipore) was applied to brain sectionsfollowed by secondary antibody conjugated with FITC. The images of NeuN⁺cells in brain sections were acquired by Nikon C2 plus confocalmicroscope with NIS image stitching function for large image capture inND acquisition. All NIS images in 32 bit were converted into 16 bitformat and analysed by ImageJ FIJI. NeuN⁺ cells were counted from wholebrain images and from multiple images (3.5×2.0 mm) in each brain region(cortex, midbrain, cerebellum and brain stem).

For cell culture immunostaining differentiated N2a cells were seeded onchamber slides and stained with anti-Map2 antibody (1:200) followed bythe protocol described above except for using 3% BSA in PBS as blockingbuffer. Fluorescent images were processed by Zeiss Apotome 200M.

Immunoblot

Half of mice brain (sagittal cut) tissues were homogenized in M-PERMammalian Protein Extraction Reagent and subjected to electrophoresis.Ryr proteins (−500 kD) in tissues lysate were separated on 3-8% NuPAGEgel running at 90V for 12 h at 4° C. in acetate buffer. 13-actin wasresolved on 4-12% Bis-Tris gel. The proteins were transferred to PVDFmembrane using iBlot 2 gel transfer device following manufacturer'sinstructions. The blots were incubated with anti-mouse Ryr3 monoclonalantibody (1/250) or anti-13-actin (1:5000) overnight at 4° C. in 1.5%BSA/1.5% milk/PBS buffer. LC3, CAMK IV and calmodulin were separated on16% Tris-glycine gel (Novex) and detected by anti-LC3 (1:1000),anti-CAMK IV (1:500) or anti-CAM (1:1000), respectively. The signalswere detected with either ECL detection reagent or AP ConjugateSubstrate Kit according to manufacturer's instructions. Band intensitieson immunoblots were quantitated by Image J (NIH, Baltimore, Va.).

Seahorse Mitochondrial Function Assay

Cultured N2a cells treated with or without CBE, dantrolene or G6 weretransferred to a XF96 assay plate at 10,000 cells/well and allowed togrow overnight in DMEM medium containing 10% FBS with each compound asdescribed above in Cell culture and treatment section. Cell numbers weredetermined using a haemocytometer. Prior to mitochondrial function assayon XF96 Extracellular Flux Analyzers (Seahorse Biosciences), the cellsin the wells of XF96 assay plates were washed by gently adding warmassay medium to the side of each well. The plates were transferred to a37° C. incubator without CO₂ for 30-60 min before the addition of XFassay medium (Seahorse Bioscience) containing 25 mM glucose and 1 mMpyruvate. After calibration of the Analyzer, the plate was sequentiallyinjected with A: 25 μl of 8 μM oligomycin, B: 25 μI of 27 μMcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and C: 25 μI of 50 μMantimycin A. Oxygen consumption rate (OCR) was measured, and the datawere analysed using the XFe Wave software. The respiration parameterswere calculated by subtracting the average respiration rates before andafter the addition of the electron transport inhibitors: oligomycin,FCCP and antimycin A. The parameters include basal respiration (baselinerespiration minus antimycin A post injection respiration), ATPproduction (baseline respiration minus oligomycin post injectionrespiration), and maximal respiratory (FCCP stimulated respiration minusantimycin A post injection respiration). Cell mitochondrial functionpresented as respiration parameters that were normalized by cell numbers(39).

Brain mitochondria were isolated from 4L;C, WT, and dantrolene treated4L;C mice as described with slight modifications (21,80). Mouse braintissues were digested with trypsin for 30 min on ice followed byhomogenization on the gentle MACS Dissociator using Programm_mito_tissues_01 (MACS, Miltenyi Biotec). Homogenates were suspended in4 ml ice-cold buffer (1.0 mM KCl, 1.0 mM Tris-HCl, and 0.1 mM EDTA, pH8.0) and mixed with 0.67 ml of 2 M sucrose. The suspension wascentrifuged at 1300×g for 5 min to remove nuclei, unbroken cells andlarge membrane fragments. The supernatant containing mitochondria werepelleted after centrifugation at 9600×g for 10 min at 4° C. Themitochondrial pellet was resuspended in the Storage Buffer (MACS,Miltenyi Biotec). Total mitochondrial protein was determined usingBradford Assay reagent (Bio-Rad). ATP production rates were determinedwith isolated mitochondria using XF96 Extracellular Flux Analyzers. Themitochondria were diluted in cold 1×MAS (Mitochondria’ Assay Solution)and substrate (pyruvate/malate) (Seahorse Biosciences). Themitochondrial suspension (20 μg mitochondrial proteins in 25 μI) wasaliquoted into each well while the plate was on ice. The plate was thencentrifuged using a swinging bucket microplate adaptor at 2000×g for 20min at 4° C. To start the assay, 155 μI of pre-warmed (37° C.) 1×MAS andsubstrate were added to each well containing isolated mitochondria andincubated at 37° C. with no CO₂ for 10 min. After calibration of theAnalyzer, the plate containing mitochondria was sequentially injectedwith A: port A, 20 μI of 40 mM ADP (4 mM, final); port B, 22 μI of 25μg/ml oligomycin (2.5 μg/ml, final); port C, 24 μI of 40 μM FCCP (4 μM,final); and port D, 26 μI of 40 μM antimycin A (4 μM, final). OCR wasmeasured and the data were analysed using the XFe Wave software asdescribed above. ATP production rate in brain mitochondria wasnormalized to mg of mitochondrial protein (18).

GCase Activity Analyses

Cells were homogenized in 1% sodium taurocholate/1% Triton X-100. GCaseactivity was determined fluorometrically using 4MU-Glucose as thesubstrate in 0.25% sodium taurocholate, 0.25% Triton X-100 and 0.1Mcitric-phosphate buffer (pH 5.6) as 5.

described previously (81). Brain tissues were homogenized in 1×PBS andincubated in 5 μM brain phosphatidylserine and 0.1M citric-phosphatebuffer (pH 5.6) for GCase activity assay using 4MU-Glucose as substrate(82). Protein concentrations were determined by BCA assay using BSA asstandard.

Glycosphingolipid Analyses

Glycosphingolipids in mouse brains and N2a cells were extracted withchloroform and methanol as described (83). GC and GS content in theextracts was analysed by ESI-LC-MS/MS using a Waters Quattro Micro APItriple quadrupole mass spectrometer (Milford, Mass.) interfaced withAcquity UPLC system as described (35). The concentration of GC and GS inthe brain was normalized to mg tissue weight and in the cells wasnormalized by mg protein in the cell lysate.

Statistical Analyses

The data are presented as mean±SEM and were analysed by Student's t-testor one-way ANOVA with post-hoc Tukey test using GraphPad Prism 6. Thelevel of significance was set at P<0.05. Survival analysis was performedusing Kaplan-Meier and the Mantel-Cox tests.

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All percentages and ratios are calculated by weight unless otherwiseindicated.

All percentages and ratios are calculated based on the total compositionunless otherwise indicated.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “20 mm” is intended to mean“about 20 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

1. A method of treating a condition associated with neuronopathicGaucher disease (nGD), in an individual in need thereof, comprisingadministering an effective amount of dantrolene or pharmaceuticallyacceptable salt thereof to said individual wherein said condition isselected from one or more of brain inflammation, neuron loss, andneurodegeneration.
 2. The method of claim 1, wherein said Gaucherdisease is type II.
 3. The method of claim 1, wherein said Gaucherdisease is type III. 4-13. (canceled)
 14. A method for a neurologicalsign in an individual having nGD, comprising administering dantrolene,or pharmaceutically salt thereof, to said individual.
 15. The method ofclaim 1, wherein said improvement in neurodegeneration is an improvementin gait impairment.
 16. The method of claim 1, wherein saidadministration is in an amount sufficient to increase GCase activity.17. The method of claim 1, wherein said administration reduces substrateaccumulation.
 18. The method of claim 1, wherein said administrationprotects calmodulin and CAMKIV expression.